Methicillin resistant S. aureus (MRSA) are leading causes of antibiotic resistant infection, treated mainly with vancomycin. Since 2002, VanA-type vancomycin resistance has moved on plasmids from vancomycin resistant enterococci (VRE) to MRSA, forming VRSA. This has happened at least 12 times independently during the course of infection, mainly in lesions on the extremities of diabetics. All S. aureus recipients of the vancomycin resistance transposon have been of a single S. aureus clade, CC5. For horizontal transmission to occur, VRE and MRSA must occupy the same environment in sufficient number and proximity for transfer to occur. I hypothesize that CC5 MRSA and VRE exhibit unique interactions compared to other MRSA lineages, which include cooperative metabolic abilities promoting synergy, co-aggregation with host-derived factors, and modulation of intra- and interspecies cell signaling. Preliminary data identified serum-dependent co-aggregation as being unique to CC5-MRSA and VRE pairs. To determine what affects multi-species binding, purified serum components will be added to co-cultures and co-aggregation will be assayed by microscopy and optical density. Any enhancement of horizontal transmission will be quantified by selective plating of MRSA recipients after co-culture with enterococci containing a resistance plasmid. I have found down-regulation of VRE genes for pheromone signaling and the fsr virulence operon in the transcriptome responses of VRE /MRSA in co-culture. The impact of cell signaling modulation will be studied by transcriptomics and targeted mutagenesis of the putative pheromone signal revealed by our microarray data. D-lactate (dLac) is commonly produced by S. aureus during fermentation. dLac is also necessary for VanA-type vancomycin resistance. As VanA type resistance is induced only when vancomycin is present, I hypothesize that S. aureus dLac production may provide an advantage to VRE in co-culture with S. aureus. This will be tested by measuring survival of both species in co- and mono-culture after vancomycin exposure as well as VRE in mono-culture incubated with dLac prior to vancomycin treatment. These results will ultimately reveal the nature of polymicrobial interactions specific to ST5-MRSA and VRE and how they lead to vancomycin resistance transfer.